This invention relates to apparatus for treating specimens, particularly biological specimens, at low temperatures for a subsequent microscopic, particularly electron microscopic, examination.
Biological specimens for microscopic, particularly electron microscopic, examinations are to an increasing extent frozen extremely rapidly (shock freezing) in order to stabilise rapidly the specimens. Afterwards the specimens may be subjected to dehydration to remove the water content, which is frequently over 90% of the weight of the specimens. This is achieved by incubating the specimens at low temperatures to exchange the water contained in the specimens for suitable organic solvents such as anhydrous acetone or methanol, or for suitable organic solutions such as OsO.sub.4 and/or uranyl acetate in anhydrous acetone or methanol. The correct control of the initial phase of this exchange is very important, and the exchange is performed at temperatures which are generally not above -80.degree. C., at least during this initial phase. If temperatures above -80.degree. C. are employed, then changes in the molecular structure of the specimens may occur, and this would depreciate the value of subsequent examination.
For safety reasons the initial phase of the dehydration generally takes place in the temperature range between -80.degree. C. and -120.degree. C., which cannot be obtained with conventional refrigeration thermostats unless there is a large outlay for apparatus.
For electron microscopy, usually 1 to 10 tissue blocks with an individual volume of between 0.1 mm.sup.3 and 10 mm.sup.3 are subjected in one operation to such a dehydration; this may take between 3 days and 3 weeks, depending upon the size of the objects and the preselected temperature. During this period the temperature must not be allowed to exceed the limit value (which has to be determined for each specimen individually) because otherwise the molecular structure of the specimens may be changed so that it is no longer possible to use the subsequent examination to make valid scientific statements about the specimen structure in the normal living state ("in vivo").
An apparatus for treating specimens is described in German Offenlegungsschrift No. 3,042,578. According to this specification the container in which dehydration and/or embedding is carried out is suspended into the neck of a Dewar vessel so that the top of the container is substantially flush with the top rim of the Dewar neck, and in this way can be easily charged with the media for dehydration and/or embedding, and with the frozen specimens.
During the dehydration and embedding, the container and the Dewar neck are covered with an insulating cover, through which only the shaft of an agitator to stir the media, and electrical connections for temperature regulation, are passed. The temperature regulation is effected by means of a heating cartridge and a temperature sensor using an electrical regulation circuit.
The Dewar vessel contains liquid nitrogen, and for incubation temperatures up to about a minimum of about -80.degree. C. cooling by cold nitrogen boiling off continuously from the liquid nitrogen is sufficient. The cooling of the container is accelerated, for the start of the work only, by a "cold finger" which is adjustable in height, and which temporarily causes a more intense thermal flux out of the container by direct metallic contact with the liquid nitrogen.
This known apparatus is substantially superior in its efficiency and its simple construction to the cooling systems employed previously.
However, certain problems arise during the charging of the container with the dehydration media and with the frozen specimens. The media employed are all extremely hygroscopic and, particularly in the cooled state, attract water very rapidly from the moist room air; this renders a low temperature dehydration of the type required impossible. There is also a possibility that the shock frozen specimens become superficially warmed above -80.degree. C. during introduction, and that irreversible heat damage occurs in the well preserved marginal zones of the specimens. This zone is frequently only 10 microns deep, and damage to it depreciates the results of the low temperature deyhydration.
Further difficulties can arise when operating with media which are employed at temperatures at or below -120.degree. C., and the dehydration is followed by an embedding which has to be performed at about -35.degree. C. An example of such embedding is a LOWICRYL low temperature embedding (cf. "Resin development for electron microscopy and an analysis of embedding at low temperature" by Carlemalm et al, 1982, Volume 126, page 123 to 143). The initial cooling to -120.degree. C. cannot be performed without a metallic connection between the container and the liquid nitrogen, but this afterwards causes an intense boiling of the liquid nitrogen to commence in the higher temperature range at which the embedding is performed, and this boiling becomes uncontrollable due the the necessary counter heating of the container.
Moreover, the multiple exchange of the media which is necessary, for example, when a low temperature dehydration is followed by a low temperature embedding, together with the necessary transfer of the dehydrated specimens into a separate chamber for the low temperature embedding, presents such substantial problems that this promising and scientifically interesting method has hitherto only rarely been adopted.